Rickettsial infection in animals and Brazilian spotted fever endemicity

We compared the rickettsial infection status of Amblyomma cajennense ticks, humans, dogs, and horses in both Brazilian spotted fever (BSF)-endemic and -nonendemic areas in the state of Silo Paulo, Brazil. Most of the horses and few dogs from BSF-endemic areas had serologic titers against Rickettsia rickettsii antigens. In contrast, no dogs or horses from BSF-nonendemic areas had serologic titers against R. rickettsii antigens, although they were continually exposed to A. cajennense ticks. All human serum samples and ticks from both areas were negative by serologic assay and polymerase chain reaction, respectively. Our results indicate that surveys of horse serum are a useful method of BSF surveillance in areas where humans are exposed to A. cajennense ticks. In addition, we successfully performed experimental infection of A. cajennense ticks with R. parkeri.
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Brazilian spotted fever (BSF) is an acute, febrile, tick-borne disease caused by the bacterium Rickettsia rickettsii. The disease is transmitted by Amblyomma ticks and has been considered endemic in some areas of the states of Sao Paulo, Minas Gerais, Rio de Janeiro, and Espirito Santo (1-7). Although the tick species Amblyomma aureolatum is the main vector of BSF in few areas of the state of Sao Paulo (8, A. Pinter, unpub data), A. cajennense is the most common tick vector associated with the disease in Brazil (9-11).

A. cajennense is a common tick in rural areas of the state of Silo Paulo, where it is also the main tick species infesting humans (12,13). In contrast, BSF cases have been reported at only a few locations within the geographic range of this tick species (14). Although unreported cases may have occurred in other areas where BSF is not known to be endemic, this possibility is unlikely for such a highly lethal disease. Ecologic differences might be the main factor regulating the occurrence of R. rickettsii among ticks and, consequently, the occurrence of the disease.

The infection rate by R. ricketlsii within a tick population can be diminished or even suppressed when a second Rickettsia species infects most of the members of that tick population (15,16). Thus, we hypothesize that the absence of human cases of BSF in some areas of the state of Sao Paulo (where human parasitism by A. cajennense is intense) is related to the presence of other, less pathogenic Rickettsia species infecting A. cajennense tick populations. In this regard, our study evaluated the rickettsial infection status of A. cajennense populations from both BSF-endemic and -nonendemic areas in the state of Sao Paulo. We also serologically evaluated humans and domestic animals from these BSF-nonendemic areas to compare it to a recent evaluation that we performed in BSF-endemic areas (17).

Materials and Methods

Study Area

The study was conducted on 6 farms in the state of Sao Paulo. Three of these farms (farms 1, 2, and 3) were considered endemic for BSF because of the recent occurrence of several laboratory-confirmed human cases of the disease among residents (4,14). These farms were the same ones evaluated in a study of Horta et al. (17). The remaining 3 farms (4, 5, and 6) were considered nonendemic for BSF because they had never had human cases of this disease. However, A. cajennense ticks were abundant there, and human infestation by this tick was a normal finding year-round among farm residents. Farms 1 (22[degrees]44'19"S, 46[degrees]55'27"W), 2 (22[degrees]47'03"S, 46[degrees]54'10"W) and 3 (22[degrees]41'14"S, 46[degrees]53'17"W) were located in the Pedreira Municipality whereas farms 4 (23[degrees]23'15"S, 47[degrees]26'14"W), 5 (23[degrees]36'43"S, 46[degrees]57'29"W), and 6 (21[degrees]57'07"S, 47[degrees]27'05"W) were located in Porto Feliz, Cotia, and Pirassununga Municipalities, respectively.

In all 6 farms, human occupations were basically divided between livestock-raising activities for men and household activities for women and children. Nevertheless, children spent substantial time in outdoor activities. All 6 farms had horses grazing on mixed overgrowth pastures, interspersed with remote forest areas. However, the major ecologic difference was large populations of free-living capybaras that inhabited livestock pastures on farms 1, 2, and 3 and the absence of this animal from horse pastures on farms 4, 5, and 6. All farms, except farm 4, had free-roaming dogs with free access to pasture and forest areas. Recent studies on ticks collected on the pastures and on horses and dogs from these 6 farms allowed the tick species A. cajennense and Dermacentor nitens to be identified on the 6 farms. In addition, the capybara tick, A. cooperi, was present on farms 1, 2, and 3 but absent in the pastures of farms 4, 5, and 6 (13,17-19). Human infestation by Amblyomma ticks was frequent on all the farms.

Ticks

From December 2000 to March 2001, free-living A. cajennense adult ticks were collected from horse pastures of the 6 farms by dragging and by using C[O.sub.2] traps. Totals of ticks collected from the farms are as follows: farm 1 (244), farm 2 (353), farm 3 (213), farm 4 (222), farm 5 (206), and farm 6 (230). All ticks were brought alive to the laboratory, where their surfaces were disinfected by immersion in 70% alcohol for 10 min followed by washing in sterile water; they were then individually tested by the hemolymph test (20). Briefly, a drop of hemolymph of each tick was dried on a glass slide and stained by the Gimenez method (21). Thereafter, ticks were frozen at -80[degrees] C until processed for DNA extraction.

DNA Extraction

All ticks were processed individually for DNA extraction. Each tick was cut into 2 symmetric halves through its median axis. One half was returned to the -80[degrees]C freezer for further studies, and the other half was used for DNA extraction according to a modification of a previously described protocol (22). For this purpose, each tick half was placed in a 1.5-mL microtube containing 150 [micro]L of TE buffer (Tris HCl 10 mmol/L, EDTA 1 mmol/L, pH 7.4) and homogenized by using a sterile micropestle. Microtubes containing the homogenized, triturated ticks were then vortexed vigorously. Next, 450 [micro]L of guanidine thiocyanate (5 mol/L) were added to the tube, which was vortexed again and incubated for 10 min at room temperature with short vortexing every 2 min. Thereafter, 100 [micro]L of chloroform was added to the tube, which was inverted several times and left resting for 2 min. The tube was centrifuged at 12,000 x g for 5 min to separate the aqueous phase, which was transferred to a clean 1.5-mL microtube. Next, 600 [micro]L of isopropanol was added to the aqueous phase (400 [micro]L), which was homogenized by inverting the tube several times and then incubated at -20[degrees]C for 2 to 18 h. Thereafter, the tube was centrifuged at 12,000 x g for 15 min; the supernatant was discarded, and the pellet was dried at room temperature and then resuspended with 30 [micro]L of buffer TE. Finally, the microtubes were incubated at 56[degrees]C for 15 min to facilitate DNA homogenization and then stored at 20[degrees]C until tested by polymerase chain reaction (PCR).

PCR

Five microliters of the extracted DNA from tick specimen was used as template for amplification of fragments of the rickettsial gltA (citrate synthase gene) and 17-kDa protein gene. A 381-bp portion of the Rickettsia gltA gene was targeted from each extracted tick DNA by using primers RPCS.877 and RpCS.1258n (23), and a 434-bp portion of the Rickettsia genus-specific 17-kDa protein gene was targeted as previously described (24). Ten microliters of the PeR product underwent electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and examined with UV transillumination. For the 10 individual ticks that were tested by PCR, a negative control (5 [micro]L of water) and positive control (5 [micrco]L of DNA extracted from an A. cajennense tick experimentally infected with R. parkeri) were included. Procedures to obtain R. parkeri experimentally infected ticks are described below. PCR results were statistically analyzed by the program @Risk Software--Risk Analysis Add-in for Microsoft Excel (Palisade Corporation, Newfield, NY, USA), which adopted Monte Carlo techniques to determine the confidence level of the prevalence of ticks infected by Rickettsia in each tick population (farm), considering [alpha] = 0.05.

R. parkeri Experimentally Infected Ticks